PROJECT SUMMARY/ABSTRACT Infections driven by extended-spectrum β-lactamase (ESBL)-producing bacteria are resistant to most or all penicillins and cephalosporins. However, there is significant risk in defaulting to carbapenems to treat any infection potentially resulting from broad- or extended-spectrum β-lactamase-producers. The use of carbapenems (a drug of last resort) may contribute to even more dangerous antimicrobial resistance and thus it is imperative to immediately and specifically confirm ESBL-based resistance in order to ensure that the selected therapeutic simultaneously minimizes risk to the patient and to the public. Current methods rely on culture of the pathogenic organisms, which requires more than 24 hours for identification. Our team aims to create a rapid (1 hr) and culture-free tool to phenotypically screen for ESBL-producing pathogens. We propose to synthesize a molecular trigger in which a cephalosporin is covalently attached to a single-stranded DNA oligonucleotide near the 3’ end. Without ESBL enzyme activity, the oligo cannot be extended by DNA polymerase; upon ESBL activity, the cephalosporin is released, enabling the DNA to be extended along DNA templates present in the assay, implying that DNA amplification can be triggered specifically by ESBL activity. Hence, we are proposing to combine the phenotypic detection of ESBL activity with the detection power of DNA amplification. The proposed approach results in an immediate discrimination of ESBL-driven resistance, thus enabling rapid treatment without sacrificing antibiotic stewardship.